Direct electron transfer (DET)-type bioelectrocatalysis, which couples the electrode reactions and catalytic functions of redox enzymes without any redox mediator, is one of the most intriguing subjects that has been studied over the past few decades in the field of bioelectrochemistry. In order to realize the DET-type bioelectrocatalysis and improve the performance, nanostructures of the electrode surface have to be carefully tuned for each enzyme. In addition, enzymes can also be tuned by the protein engineering approach for the DET-type reaction. This review summarizes the recent progresses in this field of the research while considering the importance of nanostructure of electrodes as well as redox enzymes. This review also describes the basic concepts and theoretical aspects of DET-type bioelectrocatalysis, the significance of nanostructures as scaffolds for DET-type reactions, protein engineering approaches for DET-type reactions, and concepts and facts of bidirectional DET-type reactions from a cross-disciplinary viewpoint.

Bioelectrocatalysis provides the intrinsic catalytic functions of redox enzymes to nonspecific electrode reactions and is the most important and basic concept for electrochemical biosensors. This review starts by describing fundamental characteristics of bioelectrocatalytic reactions in mediated and direct electron transfer types from a theoretical viewpoint and summarizes amperometric biosensors based on multi-enzymatic cascades and for multianalyte detection. The review also introduces prospective aspects of two new concepts of biosensors: mass-transfer-controlled (pseudo)steady-state amperometry at microelectrodes with enhanced enzymatic activity without calibration curves and potentiometric coulometry at enzyme/mediator-immobilized biosensors for absolute determination.

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)—a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD⁺)/NADH redox couples—was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize “direct electron transfer (DET)-type bioelectrocatalysis.” However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered.

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm^-2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.